Ion-exchange is one of the most useful techniques available for resolving protein mixtures. Most proteins have isoelectric points below 6 and therefore anion-exchange is more generally useful than cation-exchange. There are, however, a number of protein mixtures which are best resolved by cation-exchange.
The most widely used materials for protein cation-exchange have been cellulose or dextrans containing carboxymethyl groups. These materials exhibit the general requirements of hydrophilicity and high capacity which is necessary for general protein chromatography.
Recent advances in chromatography involving the use of finer column packings with high surface areas have led to a technique known as high performance liquid chromatography (HPLC). In modern HPLC, pressures of several thousand lb/in.sup.2 are often developed within the chromatography columns. This requires that the column packing materials be rigid and non-collapsible. The carbohydrate base materials used in protein cation-exchange heretofore are not suitable for use in HPLC as they cannot withstand the high pressures involved. To achieve materials meeting this requirements, porous inorganic materials, such as silica and alumina have heretofore been utilized as the support material with organic stationary phases on the surface to obtain a variety of liquid chromatography column packing materials. Recently, hydrophilic organic supports have been developed for this purpose, but the pressure limit of these packings is still appreciably lower than that of the inorganic base materials and their application is therefore limited. In addition these materials are not available in the wide range of pore and particle diameters that the inorganic support materials are.
Requirements for an inorganic support which is to be used for protein HPLC are that the material be microparticulate, hydrophilic and macroporous. An anion-exchange material is available which meets these requirements, but until recently no such cation-exchange materials were available. Glass is naturally anionic, and porous glass beads have been used for cation-exchange of proteins. Glass, however, is not an entirely acceptable material as it binds many proteins irreversibly. The cation exchange is best carried out through anionic groups in a hydrophilic, organic coating on the surface of the inorganic support. Such material is described in U.S. Pat. No. 4,108,603, to Chang et al. This material suffers several drawbacks in that it requires several steps to prepare and is difficult to reproduce. A more promising coating, based on polyethyleneimine-diglycolic anhydride, has been developed and support materials based on this material are commercially available.
Studies have shown the advantages of using short polymers to prepare coatings for inorganic chromatography supports. A polymer that reacts well with the support surface and not with itself will form a self-assembling coating. Such a coating is reproducible and covers the inorganic surface uniformly. The coating also tends not to fill in pores or to cement the inorganic particles together. The present invention produces a self-assembling coating which is highly reproducible and which meets the requirements of high performance liquid chromatography.